Notchl is a member of a conserved family of large modular type I transmembrane receptors that control differentiation in multicellular animals. Notch function is mediated through a novel signal transduction pathway involving successive ligand-induced proteolytic cleavages that serve to release the intracellular domain of Notch, which then translocates to the nucleus and activates downstream transcription factors. The extracellular domain of all Notch receptors have three iterated LIN 12 modules that appear to act as negative regulatory domains, possibly by limiting proteolysis. Each LIN 12 module contains three disulfide bonds and three conserved aspartate (D) or asparagine (N) residues. To begin to understand the structural basis for LIN12 function, the first LIN12 module of human Notchl (rLIN12.1) has been expressed recombinantly in E. coli and purified in reduced form. In redox buffers, rLIN12.1 forms only one disulfide isomer in the presence of millimolar Ca++, whereas multiple disulfide isomers are observed in the presence of Mg ,-, and EDTA. One-dimensional proton nuclear magnetic resonance shows that Ca + induces a dramatic increase in chemical shift dispersion of the native rLIN12.1 amide protons, as seen for Ca ++ -binding LDL-A modules. We have identified conditions suitable for determining the solution structure of rLIN 12. 1 by NMR. Using recombinant '5N-labelled protein, we have completed backbone assignments of rLIN12.1 and side-chain assignments are in progress. In addition, we have successfully refolded a recombinant fragment of Notchl that spans all three LIN12 modules and intend to pursue solution structural studies of the entire LIN12 domain.